Antibiotic A-26771 factors and process for production thereof

ABSTRACT

Antibiotic mixture A-26771, comprising microbiologically active factors A, B and C, produced by fermentation of Penicillium turbatum Westling NRRL 5630, and isolation and separation of the individual factors thereof by extraction, chromatography and crystallization. The A-26771 mixture and factors A, B and C thereof have antifungal and broad-spectrum antibacterial activity; factors A and C also have antiviral activity.

This is a division of application Ser. No. 298,053, filed Oct. 16, 1972,now U.S. Pat. No. 3,883,561.

SUMMARY OF THE INVENTION

This invention relates to antibiotic substances. In particular, itrelates to a group of heterocyclic antibiotic substances which areproduced by culturing a hitherto undescribed strain of the organismPenicillium turbatum NRRL 5360.

The antibiotics of this invention are arbitrarily designated herein asA-26771 antibiotics. Three individual antibiotics have been isolated,separated and characterized from the antibiotic A-26771-producingculture. These individual antibiotics are designated as A-26771 factorsA, B and C.

The A-26771 group of antibiotics is produced by culturing a novel strainof Penicillium turbatum under submerged aerobic fermentation conditionsuntil a substantial level of antibiotic activity is produced. TheA-26771 antibiotics are recovered by extraction of both the broth andthe mycelia with polar organic solvents. Extraction of the broth andevaporation of the organic solvent give a mixture of A-26771 antibioticswhich contains mainly factor A and a small quantity of factor C.Extraction of the mycelia with organic solvent and evaporation of thissolvent give an additional amount of A-26771 antibiotics in which factorB is the main component.

The individual A-26771 antibiotics have been separated from each otherand isolated as individual antibiotic compounds by columnchromatography, thin-layer chromatography and crystallizationprocedures.

The A-26771 antibiotics inhibit the growth of organisms which arepathogenic to animal and plant life, including gram-positive andgram-negative bacteria and various fungi. In addition, factors A and Chave antiviral activity.

DESCRIPTION OF THE DRAWINGS

The infrared absorption spectrum for each of the A-26771 antiobioticfactors is presented in the drawings as follows:

FIG. 1 -- Factor A;

FIG. 2 -- Factor B;

FIG. 3 -- Factor C.

DETAILED DESCRIPTION OF THE INVENTION

The A-26771 antibiotic factors A, B and C are produced by cultivatingunder controlled conditions a hitherto undescribed strain of Penicilliumturbatum NRRL 5630.

As is the case with many antibiotic-producing cultures, fermentation ofan antibiotic A-26771-producing strain of Penicillium turbatum resultsin the production of a number of antibiotic substances. AntibioticA-26771 factors A, B, and C are three of these substances. Othersubstances are present in only very minor quantities or are relativelyunstable.

The antibiotic factors co-produced during the fermentation are obtainedas a mixture and are separated from each other and isolated asindividual compounds as hereinafter described.

The following paragraphs describe the physical and spectral propertiesof the various A-26771 factors which have been characterized.

Antibiotic A-26771 factor A is a crystalline compound (yellow platesfrom acetone) which has a melting point of about 105°C. The molecularweight of factor A, as determined by mass spectrometry, is 324.Elemental analysis of factor A gave the following percentagecomposition: carbon, 52.1%; hydrogen, 5.05%; nitrogen, 8.1%; oxygen,15.1%; sulfur, 19.4%. The calculated empirical formula is C₁₄ H₁₆ N₂ O₃S₂. The observed specific rotation of factor A, [α]_(D) ²⁵, is -88° (c =0.15, methanol).

The infrared absorption spectrum of antibiotic A-26771 factor A inchloroform is shown in FIG. 1 of the accompanying drawing. Thedistinguishable bands in the infrared spectrum are as follow: 2.83,3.34, 5.95 (very strong), 6.68, 6.88, 7.09, 7.44, 7.96, 9.04, 9.38,9.65, and 4.40 microns.

The ultraviolet spectrum of factor A showed only end absorption.Electrometric titration of factor A in 66 percent dimethylformamide didnot indicate the presence of titratable groups.

The nuclear magnetic resonance (NMR) spectrum of A-26771 factor A inchloroform showed the following characteristics: δ 7.34 (5H, singlet);4.12, 3.62 (2H, AB, quartet, J = 16 Hz); 3.5-3.7 (1H multiplet,exchangeable); 4.3-4.5 (2H, multiplet), 3.21 (3H, singlet); and 2.98 ppm(3H, singlet).

Factor A is soluble in common polar organic solvents such as ethylacetate, ethanol, methanol, chloroform and the like, but is relativelyinsoluble in water.

Based on the various foregoing physical characteristics, the structureof antibiotic A-26771 factor A has been determined to be as shown inFormula I: ##SPC1##

which structure is designated3-benzyl-6-(hydroxymethyl)-1,4-dimethyl-3,6-epidithio-2,5-piperazinedione.

Antibiotic A-26771 factor B is a white crsytalline compound (longneedles from acetone-water), melting at about 125°C. As determined bymass spectrometry, the molecular weight of factor B is 382. Elementalanalysis of factor B gave the following percentage composition: carbon,62.9%; hydrogen, 7.7%; oxygen, 29.0%. The calculated empirical formulais C₂₀ H₃₀ O₇. The observed specific rotation of factor B, [α]_(D) ²⁴,is -14° (c = 0.13, methanol).

The infrared absorption spectrum of antibiotic A-26771 factor B inchloroform is shown in FIG. 2 of the accompanying drawings. Thedistinguishable bands in the infrared spectrum are as follows: 3.30,3.40, 3.50, 5.86 (very strong), 6.20, 6.87, 7.26, 7.40, 7.70, 7.82,8.65, 8.90 and 10.26 microns.

Factor B absorbs in the ultraviolet region of the spectrum and exhibitsabsorption maxima in both neutral and acidic ethanol solutions at λ max.222 mμ (ε 16,000) which shifts to λ max. 365 mμ (ε 6,000) in base. Theshift is irreversible.

The NMR spectrum of A-26771 factor B in chloroform showed the followingcharacteristics: δ 9.30 (1H, singlet); 7.25, 6.75 (2H, quartet, AB, J =16 Hz); 5.0-5.5 (2H, multiplet); 2.73 (4H, singlet); and 1.0-2.0 ppm(21H, multiplet).

Factor B is slightly soluble in polar organic solvents such as ethylacetate, chloroform, acetone, ethanol, methanol, and the like, but isrelatively insoluble in solvents such as water and diethyl ether.

Antibiotic A-26771 factor B is an ionophorous agent and, as such,inhibits glutamate oxidation in rat-liver mitochondria. The antibioticmay produce this effect by interfering with the transport of calcium ordivalent cations.

Based on the various observed physical characteristics, a proposedstructure of antibiotic A-26771 factor B has been devised. The structurehas not been determined with certitude, however, and it is to beunderstood that the structure presented herein represents merely aworking hypothesis. The structure postulated for factor B is as shown inFormula II: ##SPC2##

which structure is designatedmono(16-methyl-2,5-dioxooxacyclohexadec-3-en-6-yl) succinate.

Antibiotic A-26771 factor B is capable of forming ester derivatives bystandard procedures, for example, by treatment with alcohol in thepresence of an acid catalyst. These ester derivatives are also useful asantibiotics.

Antibiotic A-26771 factor C is a crystalline compound (colorless prismsfrom acetone-ether-pentane) melting at about 130°C. The molecular weightof factor C is 388, as determined by mass spectrometry. Elementalanalysis of factor C gave the following percentage composition: carbon,43.12%; hydrogen, 4.21%; nitrogen, 7.02%; sulfur, 32.5%. The calculatedempirical formula is C₁₄ H₁₆ N₂ O₃ S₄. The observed specific rotation offactor C, [α]_(D) ²⁵, is -187° (c = 0.04, methanol).

The infrared spectrum of antibiotic A-26771 factor C in chloroform isshown in FIG. 3 of the accompanying drawings. The distinguishable bandsin the infrared spectrum are as follows: 2.79, 3.32, 6.00, 6.68, 6.89,7.09, 7.30, 7.88, 9.10, 9.40, 9.60 and 14.45 microns.

The ultraviolet absorption spectrum of factor C in acidic ethanol showsan absorption maximum at λ max. 275 mμ(ε 10,000). In neutral solution,the UV spectrum of factor C shows only end absorption with a plateau inthe 275-300 mμ range (molar absorption about 1200); in basic solutionthis plateau shifts to a shoulder at 260 mμ (molar absorptionapproximately 7,000) and a plateau at about 290-300 mμ (molar absorptionabout 4,000).

The NMR spectrum of A-26771 factor C in chloroform-D₂ O showed thefollowing characteristics: δ 7.26 (5H, multiplet); 4.26, 3.85 (2H, AB,quartet, J = 13 Hz); 4.05, 3.25 (2H, AB, quartet, J = 15 Hz); 3.13 (3H,singlet); and 3.07 ppm (3H, singlet).

Factor C is soluble in common polar organic solvents such as ethylacetate, ethanol, methanol, chloroform, and the like, but is relativelyinsoluble in water.

Based on the physical characteristics hereinabove recited, a proposedstructure for antibiotic A-26771 factor C can be set forth. Since thestructure determination is merely postulated, however, it is to beunderstood that the structure presented herein represents merely aworking hypothesis. The tentative structure for factor C is shown inFormula III: ##SPC3##

which structure is designated3-benzyl-6-(hydroxymethyl)-1,4-dimethyl-3,6-epitetrathio-2,5-piperazinedione.

The mixture of A-26771 antibiotics and the individual factors thereofinhibit the growth of microorganisms which are pathogenic to animal andplant life, including gram-positive and gram-negative bacteria andvarious fungi. In addition, factors A and C are antiviral agents.

In Table I which follows, the inhibition-zone diameters in millimetersof the A-26771 antibiotic factors, as determined by the standarddisc-plate assay, are listed for several illustrative microorganisms.

                                      TABLE I                                     __________________________________________________________________________    Disc-Plate Activity of Factors A, B, and C                                                        Factor A                                                                              Factor B                                                                              Factor C                                                 mg./ml.                                                                            1.0 0.1 1.0 0.1 1.0 0.1                                   __________________________________________________________________________    Organism                                                                      Staphylococcus aureus                                                                             23  12  24  15  20  --                                    Mycobacterium avium 14   tr*                                                                               9  --  11  --                                    Bacillus subtilis   22  11  14  tr  20  tr                                    Sarcina lutea       28  18  22  12  26  14                                    Escherichia coli    12  --  --  --  tr  --                                    Klebsiella pneumoniae                                                                             12  --  --  --  tr  --                                    Serratia marcescens 14  --  --  --  14  --                                    Pseudomonas solanacearum                                                                          13  --  --  --  --  --                                    Saccharomyces pastorianum                                                                         32  19  25  12  25  --                                    Neurospora crassa   27  13  13  --  22  --                                    Candida albicans    28  tr  tr  --  23  --                                    Fusarium moniliforme                                                                              14  10  tr  --   8  --                                    Trichophyton mentagrophytes                                                                       27  19  14  tr  19  12                                    __________________________________________________________________________     *trace?                                                                  

The in vitro susceptibilities of selected bacteria to antibiotic A-26771factor B have been determined by standard broth-dilution andagar-dilution assays. The minimal inhibitory concentrations (MIC), asdetermined by these methods, are summarized in Table II.

                  TABLE II                                                        ______________________________________                                        In vitro Activity of A-26771 Factor B                                         Organism               MIC (μg./ml.)                                       ______________________________________                                        Organism                                                                      Staphylococcus aureus.sup.a                                                                          <1.56                                                  Streptococcus faecalis.sup.a                                                                         25.0                                                   Vibrio coli.sup.b      50.0                                                   Mycoplasma gallisepticum.sup.b                                                                       3.12                                                   Mycoplasma granularum.sup.b                                                                          50.0                                                   Mycoplasma synoviae.sup.b                                                                            12.5                                                   Mycoplasma hyosynoviae.sup.b                                                                         12.5                                                   Mycoplasma hyopneumoniae.sup.b                                                                       12.5                                                   Erwinia amylovora.sup.a                                                                              <0.78                                                  Pasteurella multocida.sup.b                                                                          6.25                                                   Xanthomonas phaseoli.sup.a                                                                           50.0                                                   Candida tropicalis.sup.a                                                                             100.0                                                  Trichophyton mentagrophytes                                                                          6.25                                                   Botrytis cinerea       12.5                                                   Ceratocystis ulmi      6.25                                                   Verticillium albo-atrum                                                                              50.0                                                   ______________________________________                                         .sup.a agar dilution?                                                         .sup.b broth dilution?                                                   

The activity of antibiotic A-26771 factors A and C against viral growthin tissue culture has been demonstrated against several viruses,including polioviruses and Coxsackie virus. In addition, the antiviralactivity of factor A has been demonstrated in vivo from the biologicalactivity of serum levels in mice.

The ability of antibiotic factors A and C to control the growth of virusin vitro is readily demonstrated by using a plaque suppression testsimilar to that described by Siminoff, Applied Microbiology, 9 [1],66-72 (1961). The test is carried out as follows:

Rectangular glass boxes measuring 71/2 × 15 × 11/2 inches, made ofpieces of double-strength plate glass sealed together with siliconerubber cement, are used in carrying out the test. The glass boxes arecovered with a glass lid and, before use, are sterilized by dry heat ata temperature of about 300° C. Approximately 10⁶ /ml. BS-C-1 serialculture of Cercopithecus monkey kidney, Hopps et al.) cell suspension ismade in a medium composed of tissue culture medium 199, 5 percent ofcalf serum, penicillin (150 units/ml.) and streptomycin (150 μg./ml.). Aportion of this suspension (250 ml.) is added to each sterilized glassbox, and the box is incubated at about 37°C. for about 96 hours in alevel position. After incubation, the medium is carefully drawn off,leaving a monolayer of cells undisturbed on the glass. The cells arethen infected by gently adding to each box about 100 ml. of a suspensionof virus in medium 199.

After a time of approximately 1 to 3 hours is allowed for adsorption ofthe virus on the cells, the infecting medium is removed from the plate.A mixture of 75 ml. of double-strength medium 199 with calf serum,penicillin, and streptomycin and 75 ml. of double-strength agar solution(Difco, purified, 2 percent) at 50°C. is poured over the virus-infectedcell monolayer in each box and allowed to solidify at a level attitude.Filter-paper disks are dipped in solutions of the test substances, driedin a vacuum oven at no higher than 37°C. for about 1 hour, and thenplaced on the surface of the agar in the boxes. The boxes are incubatedat about 37°C. for about 84 hour and are then flooded with aqueous 10percent formalin-2 percent sodium acetate solution, and the agar isfloated off with water. The cells remaining are stained with Wright'sstain. Toxicity, antiviral activity, and the microscopic appearance ofthe cells are recorded following the procedure of Herrmann et al., Proc.Soc. Exp. Biol, Med., 103, 625 (1960).

Plaques are seen in those areas of the plate where the virus hasinfected and reproduced in the cells. Zones of toxicity are alsoobserved, and the diameters thereof are measured in millimeters wheneverthe test compound kills the cells under and around the filter-paperdisk. Antiviral activity of the test compounds is detected by observingthe absence of plaques and the presence of a heavier growth of cells ina zone under and around the filter-paper disks.

The cells in a zone of activity are examined with a microscope todetermine the presence and degree of drug and/or virus damage. Thestaining is graded 1+, 2+, 3+, 4+, and negative to reflect thefollowing:

4+ dark stained areas which, upon microscopic examination, show healthycells with no visible virus or drug damage;

3+ less darkly stained areas that show no virus or drug damage butappear less healthy;

2+ areas showing healthy cells with a moderate amount of virusbreakthrough;

1+ areas showing healthy cells with a greater virus breakthrough;

- no viable cells.

Table III which follows sets forth the results of tissue-culture test ofantibiotic A-26771 factors A and C against Type III poliovirus andCoxsackie virus A-21 (COE strain). In the table, column 3 gives thediameter in millimeters of the zone of virus inhibition by the testcompound, anad column 4 indicates the grading of the stained areas.

                  TABLE III                                                       ______________________________________                                        In Vitro Antiviral Activity of Factors A and C                                       Conc.     Zone    Microscopic                                                 μg./ml.                                                                              mm.     Examination Virus                                    ______________________________________                                        Factor A 1000        50      4 +       polio                                           500         50      4 +       polio                                           250         46      4 +       polio                                           125         44      4 +       polio                                           62.2        40      4 +       polio                                           31.1        38      4 +       polio                                           15          34      4 +       polio                                           7.5         30      4 +       polio                                           3.2         24      4 +       polio                                           1.5         16      4 +       polio                                  Factor C 1000.0      50      4 +       polio                                           100.0       40      4 +       polio                                  Factor A 1000.0      52      4 +       COE                                             500.0       50      4 +       COE                                             250.0       45      4 +       COE                                             125.0       40      4 +       COE                                             62.2        38      4 +       COE                                             31.1        35      4 +       COE                                             15.0        32      4 +       COE                                             7.5         26      4 +       COE                                             3.2         20      4 +       COE                                             1.5         15      4 +       COE                                    Factor C 1000.0      50      4 +       COE                                             100.0       40      4 +       COE                                    ______________________________________                                    

The ability of antibiotic A-26771 factor A to control the growth ofvirus is further demonstrated by antiviral testing in mice, using aprocedure as follows:

White Swiss mice (11-13 g.) were divided into groups of four mice each.Each group was given an intraperitoneal injection of drug in differingconcentrations, including a control group treated only with carriers.One hour after injection of the drug, all mice were bled from theorbital sinuses. Repeat bleedings from mice surviving acute toxicitywere made at six hours post-injection. Serums were separated fromcollected blood by spinning at 11,000 rpm in a microhematocritcentrifuge.

Cells susceptible to poliovirus III (BSC₁) were grown to confluency onassay plates and were then infected with poliovirus III. After a periodof adsorption the plates were overlayed with agar. One-fourth-inch fiberpads were impregnated with 20 μl. of mouse serum. The pads werepositioned on the surface of the agar-overlayed infected plate. Theplates were incubated for 72 hours at 37°C. and then were fixed with 10%formalin--2% sodium acetate for 1 hour, after which the agar was floatedoff in flowing tap water. The plates were then stained with tetrachromestain (MacNeal). If biologically active levels of compounds are present,a zone of viral inhibition occurs at the positions of the padscontaining serum from treated animals. No zones appear at the positionsof pads containing serum from untreated animals.

The results of this test are summarized in Table IV (Z indicates zonediameter in millimeters; M indicates grading of stained areas as definedhereinabove):

                  TABLE IV                                                        ______________________________________                                        Antiviral Activity of Factor A in Mouse Serum                                 One Hour             Six Hours                                                Dosage                                                                        mg./kg. Survivors Z      M     Survivors                                                                             Z    M                                 ______________________________________                                        234     4         30     4 +   0                                              117     4         29     4 +   0                                              58      4         24     4 +   4       23   4 +                               29      4         20     4 +   4       18   4 +                               ______________________________________                                    

In some instances mixture of the A-26771 antibiotics can be employedwithout the necessity of separating and using the individual factors.For example, in fighting fungal growth on environmental surfaces such asshower stalls, walls, floors, tables and the like, a mixture of theantibiotics is suitable. When used in combatting infections in aninfected host however, it is desirable to employ a single factor, forexample factor B, for administration.

As antibacterials and antifungals, the A-26771 antibiotics are usefulfor incorporation into topical preparations. Such preparations areadapted for administration to subjects exposed to, or infected with,sensitive bacteria or fungi. Suitable preparations include, for example,ointments, creams, emulsions, salves, emollients and sprays. Althoughthe optimal concentrations will differ dependent upon variables such asthe infecting bacteria or fungi, the factor or mixture of factorsemployed, and the like, the A-26771 antibiotics are typically effectivein topical preparations in concentrations of from about 0.05 to about2.0 percent by weight.

In addition, effective amounts of the A-26771 antibiotics may beincorporated into various pharmaceutical formulations, such assuspensions, emulsions, powders, dusts, soaps, detergents, aerosols andthe like, for purposes of cleaning, disinfecting, or sterilizing. Thus,for example, the A-26771 antibiotics may be used to disinfect surfacessuch as animal-body surfaces, dental and surgical instruments, glasswarewalls, floors, tables and the like. For purposes such as cleaning,disinfecting or sterilizing, the A-26771 antibiotics are effective inconcentrations as low as 0.1 percent by weight.

Furthermore, antibiotic A-26771 factors A and C are also antiviralagents. Thus, solutions containing either of these factors, preferablytogether with a surfactant, can be used to decontaminate the in vitrohabitat on which viruses, such as polio or Coxsackie, are present.Solutions containing factors A or C are effective in the control ofviruses when concentrations of factors A or C ranging from about 1 toabout 1500 μg./ml. or more are used.

In another aspect of the invention, the A-26771 factors A and C can beadministered parenterally to mammals for the control of viruses. Usefuldosage levels for prevention or treatment of viral diseases vary fromabout 5 to about 60 mg./kg. of mammalian body weight, depending uponwhether the drug is to be used prophylactically or therapeutically.

The newly-found and hitherto undescribed organism strain which producesthe A-26771 antibiotics was isolated by standard serial-dilutionprocedure from a soil sample collected from Mt. Ararat in EasternTurkey. The organism has been taxonomically characterized as a newstrain of Penicillium turbatum Westling which is in the Penicilliumthomii series, Section Monoverticillata.

A culture of the A-26771-producing organism has been deposited with thepermanent culture collection of the Agricultural Research Service,Northern Utilization Research and Development division, U.S. Departmentof Agriculture, Peoria, Ill. 61604, where it has been deposited withoutrestriction as to availability and has been accorded the accessionnumber NRRL 5630.

The characteristics of Penicillium turbatum NRRL 5630 are given in thefollowing paragraphs.

Color names were assigned according to the ISCC-NBS method described byKelly et al. in "The ISCC-NBS Method of Designating Colors and aDictionary of Color Names," National Bureau of Standards Circular 553,Washington, D.C., U.S. Government Printing Office. Color shades (M&P)are designated using "A Dictionary of Color" by A. Maerz and M. ReaPaul, McGraw-Hill, New York, N.Y.

CULTURAL CHARACTERISTICS Potato-Dextrose Agar.

Colonies are velutinous. A slightly crenated, nearly white marginal areaborders colonies which are light grayish olive (ISCC-NBS 109), quakergray -(M&P 21-B-1). The reverse color is oyster gray (M&P 19-A-1), paleyellow green (ISCC-NBS 121). The colonies achieve 40 mm. in seven dayswith little further growth when incubated at 26°-28°C. Sclerotia areformed in 7 days and are at first hyaline, then yellow-brown, globose tosubglobose, comparatively soft; and when crushed, their outer wallappears constructed of pseudoparenchymatous cells. They measure 94-320 μand average 219 μ.

Malt-Extract Agar

Colonies up to 40 mm. in diameter are produced in seven days. Earlysporulation occurs in 4 days and becomes relatively heavy in 5 days;however, relatively few penicilli are formed. The colony surface issmooth, a gray-green shade with a 5-mm. periphery of white--mermaid (M&P21-B-6) which is grayish yellow-green (ISCC-NBS 122). The reverse coloris oyster white (M&P 10-A-1), pale yellow-green (ISCC-NBS 121).

No sclerotia are observed at 2 weeks. After 3 weeks some scatteredsclerotia appear which are similar to those described on potato-dextroseagar. They measure 85-240 μ and average 190 μ. Conidia are spherical tosubglobose, smooth, dry, hyaline microscopically, greenish en masse, inbasipetal chains and range from 3.0-5.0 μ, averaging 3.4 μ. Sterigmataare generally observed in groups of 3, occasionally 5. They are slightlytapered near their apex and are smooth-walled. They are not supported byverticils of metulae or branches but occur as terminal adornment onconidiophores. Occasional aberrant forms are elaborated, such as asecond penicillus formed terminally on the central sterigma of theverticil which is associated with the conidiophore. Single terminal orlateral phialides are rarely formed. Sterigmata range from 7-15 μ longand 1.4 to 3.0 μ wide, averaging 13 82 to ×to 2.8 μ. Some branches arestrongly divaricate from the main conidiophore and are, therefore,considered monoverticillate. Branches are up to 60 μ long. Mainconidiophores are 2-4 μ and 65-108 μ long.

Czapek's-Solution Agar.

Colonies achieve 35 mm. in diameter in seven days with no further changein size. The colony is strongly wrinkled with deep furrows which radiatefrom a highly wrinkled center. It appears to be a thin velvety felt thatis not so heavily sporulated as when grown on malt-extract agar orpotato-dextrose agar. After seven days the colony surface is ivory white(M&P 10-B-2) or pale yellow (ISCC-NBS 89). After 12 days the surfacebecomes oyster white (M&P 10-B-1) or pale yellow-green (ISCC-NBS 121).The reverse surface is white jade (M&P 10-A-2), pale yellowish-pink(ISCC-NBS 31). There is neither describable odor nor exudate. Sclerotiaare not present in 2 weeks. A very few sclerotia appear in 3 weeks.These sclerotia appear to cause a reddish-brown color in their immediatevicinity. They range in size from 175-406 μ and average 349 μ.

MORPHOLOGICAL CHARACTERISTICS

This culture produces single verticils of sterigmata at the tips ofconidiophores which arise from the agar or from rambling hyphae. Somebranches occur which appear as individual conidiophores terminating withverticils of sterigmata. These sterigmata (3-5) give rise to chains ofconidia. Each branch appears to be monoverticillate. No branching occursat the apex of the main axis.

A relationship is seen with the biverticillate Asymetrica-Divaricata,specifically Penicillium pulvillorum in the P. raistrickii series.However, P. pulvillorum produces rough-walled conidiophores andsterigmata as well as definite metulae, also rough-walled; P. turbatumhas smooth-walled conidiophores and sterigmata with no definite metulae.Furthermore, P. pulvillorum colonies grown on malt-extract agar arereddish shades on the reverse side. The fact that P. pulvillorumproduces abundant sclerotium on Czapek's-solution agar does not conformwith the literature description of Penicillium turbatum.

Based on the foregoing taxonomic description of the A-26771-producingstrain, the organism has been classified as a novel strain ofPenicillium turbatum Westling. The A-26771-producing organism differsslightly from Penicillium turbatum Westling as described by Raper andThom in the "Manual of the Penicillia." The greatest difference occursin sclerotia production, which in the published description is reportedas heavy on malt-extract agar and non-existent on Czapek's-solutionagar. Sterigmata of the A-26771-producing strain are a little largerthan those described by Raper and Thom. These differences, however, aremerely strain differences; in all other respects the characteristics arein agreement with the published description of that species.

The culture medium employed to produce the antibiotic mixture can be anyone of a number of media since the A-26771-producing organism is capableof utilizing energy from a variety of nutrient sources. Thus, forexample, a variety of carbohydrates can be included in the culturemedium to supply the carbon requirements for the organism. Likewise,various sources of nitrogen, such as distillers' extracts, amino acids,and like nitrogen sources can be employed in the A-26771 medium. Forreasons of economy in production, optimal yield, and ease of isolationof the antibiotic, certain culture media are preferred. The preferredsources of carbohydrate in the fermentation, for example, are glucoseand sucrose, although other sugars can also be used. Preferred sourcesof nitrogen are peptones, but soybean meal, amino acid mixtures, and thelike are also useful. As is common in the production of antibiotics ingeneral, nutrient inorganic salts can be incorporated in the culturemedium for the production of A-26771 antibiotics. Such inorganicnutrients are the customary salts capable of yielding sodium, potassium,ammonium, calcium, phosphate, chloride, carbonate, sulfate and likeions.

Minor elements necessary for optimum growth and development of theA-26771-producing organism can also be included in the culture medium.Such trace elements commonly occur as impurities in the otherconstituents of the medium in amounts sufficient to meet the growthrequirements of the A-26771-producing organism.

The strain of the organism employed to produce the A-26771 antibioticsis capable of growing under a variety of conditions. The organism willgrow, for example, in a variety of media in which the initial pH variesrather extensively. It has been found desirable, however, to initiatethe fermentation in a medium at a pH between about 6 and about 8 andpreferably between about 6.5 and 7.0. Commonly, during the growth of theorganism, the pH of the medium will decrease gradually from the initiallevels to a pH of about 4 or lower. The final pH of the culture mediumis dependent, at least in part, on factors such as the initial pH of themedium, the buffer present therein, and the length of time the orgnismis allowed to grow.

The A-26771-producing organism of the present invention is capable ofgrowing and producing antibiotic activity on various types of media. Theorganism can be grown, for example, on agar slants, in shake flasks, orin medium- or large-scale fermentation tanks. For large-scale productionof the antibiotics of the present invention, submerged aerobicfermentation conditions are preferable. For the preparation ofrelatively small quantities of the A-26771 antibiotics, shake-flask orsurface culture in bottles can be employed.

In carrying out the submerged aerobic fermentation for production of theA-26771 antibiotics, the organism is first grown on agar-slant cultures;and the spores of the fungus are transferred from the slant to avegetative medium. The vegetative medium thus inoculated with sporesfrom the agar slant insures a rapid growth of the organism on a smallscale. The inoculated vegetative medium is allowed to grow until aviable culture of the growing organism is obtained; it is then used toinoculate the larger-scale tank. Fermentation media in large-scaletanks, thus inoculated by means of a vegetative medium, provide morerapid growth of the organism than that obtained by direct inoculationwith spores from an agar slant. Such rapid growth obtained with avegetative inoculum is highly desirable from the production standpointin that fermentation time and overall production time are reduced.

The strain of the organism producing the A-26771 antibiotics grows bestat temperatures between about 20 and 30°C. Optimal production of theantibiotic appears to occur at temperatures between about 24° and about28°C.

As in other aerobic, submerged culture processes, sterile air is blownthrough the culture medium during the fermentation period. For efficientgrowth of the organism and consequent efficient production of theantibiotic, the volume of air employed is preferably in excess of about0.1 volume of air per volume of culture medium per minute. In thesubmerged aerobic fermentation of the A-26771 antibiotics, optimalyields are obtained when the volume of air employed is at least 0.3volumes of air per volume of culture medium per minute.

In genral, maximum production of the A-26771 antibiotics occurs betweenabout 3 and about 10 days after the inoculation of the culture mediumwhen submerged aerobic fermentation conditions or shake-flask cultureconditions are employed. The maximum production of the antibiotics inlarge-scale fermentation tanks under submerged aerobic conditions isreached in about 8 days.

The course of the fermentation can be followed by assaying thefermentation medium from time to time against an organism susceptible tothe A-26771 antibiotics. Three such organisms which can be employed areSarcina lutea, Staphylococcus aureus, and Bacillus subtilis.

Following the production of the antibiotics under submerged aerobicfermentation conditions, the A-26771 antibiotic mixture previouslydescribed can be recovered from the fermentation medium by methodscommonly employed in the fermentation art. The majority of factor Bproduced is found associated with the mycelium. The majority of factorsA and C produced is found in the filtered broth.

Under the conditions employed thus far, the strain of the organismdescribed previously and designated as NRRL 5630 produces antibioticfactor B as the predominant antibiotic factor. In general, factor Baccounts for between about 85 and 95% of the total recovered antibioticactivity. The remaining factors account for substantially the remainderof the antibiotic activity.

The A-26771 antibiotics are recovered from the fermentaion medium asantibiotic mixtures. Since the majority of the antibiotic activity isassociated with the mycelia, the fermentation medium if filtered; andthe mycelia are extracted with a suitable organic solvent. To obtainfactors A and C, which are present in greatest quantity in the broth,the filtered broth is also extracted with a suitable organic solvent.

Solvents which are esters, such as ethyl acetate, amyl acetate andisoamyl acetate are suitable solvents useful for extracting either themycelia or the filtered broth. Ethyl acetate is a particularly desirableextractant. The extracts are then dried and evaporated to dryness toafford the crude antibiotic mixtures.

Alternatively, the whole fermentation broth can be extracted with asuitable organic water-immiscible solvent, such as ethyl acetate, andthereafter filtered. The extract is treated as in the first instance toobtain the crude antibiotic mixture.

The crude mixtures of A-26771 antibiotics are purified by methods suchas fractional crystallization of the mycelial extract or chromatographyof the broth extract over a suitable adsorbent such as silica gel oralumina.

In a preferred recovery procedure, the mycelia and broth are separatedby filtration. Subsequently, the separated mycelia are extracted with asuitable organic solvent such as ethyl acetate or amyl acetate; and theextract is dried and concentrated to afford a crude antibiotic mixturewhich contains factor B as a major component. In addition, the filteredbroth is also extracted with a suitable organic solvent such as ethylacetate; this extract is dried and evaporated under vacuum to give anantibiotic mixture which contains larger quantities of factors A and C.

The individual antibiotic factors can be separated from each other andisolated as individual antibiotics by techniques known in the art. Thecrude mixtures, for example, can be separated by chromatography oversilica or alumina. Alternatively, individual antibiotics can be obtainedby fractional crystallization.

Because the majority of the most abundant factor, factor B, is presentin the mycelia, it is possible to separate and isolate this factor bycrystallization techniques alone. Thus, factor B is obtained incrystalline form by concentrating the dried ethyl acetate extract of themycelia to a small volume from which a large portion of factor Bprecipitates. Cooling the concentrated solution facilitatescrystallization. The precipitate is filtered and purified further byrecrystallization.

The other described factors which occur individually as minor antibioticfactors are more difficult to separate from one another. In order toobtain factors A and C, a combination of chromatographic separations andcrystallization techniques is required. Accordingly, the dried ethylacetate extract from the broth is concentrated to an oil which is thenchromatographed, preferably over silica gel, employing solvent mixturessuch as ethyl acetatemethanol to effect a partial separation of theremaining factors. Further purification is achieved by subsequentchromatographic separations of the eluate fractions from the silica gelchromatogram and by fractional crystallizations to provide factors A andC.

The following examples are provided in order to more fully illustratethe methods and procedures of the present invention.

EXAMPLE 1 Production of the Antibiotic A-26771 Mixture

Spores of Penicillium turbatum strain NRRL 5630 were inoculated on anutrient agar slant having the following composition:

    Ingredient           Weight/Volume (g./l.)                                    ______________________________________                                        Glucose              20.                                                      Peptone              5.                                                       Potassium dihydrogen phosphate                                                                     0.5                                                      Magnesium sulfate    0.02                                                     Ferrous sulfate      0.01                                                     Agar                 20.                                                      Deionized water      g.s. 1 liter                                             ______________________________________                                    

The inoculated agar slant was incubated for 7 days at a temperature of25°C. and refrigerated until used. The agar slant was then covered withbeef serum and was gently scraped with a sterile needle to suspend thespores and cells in the serum. The resulting suspension was divided intosix tubes for lyophilization. One of the lyophilized pellets thusprepared was used to inoculate 100 ml. of a vegetative medium having thefollowing composition:

    Ingredient           Weight/Volume (g./1.)                                    ______________________________________                                        Sucrose              25.                                                      Edible molasses      36.                                                      Corn Steep           6.                                                       Potassium dihydrogen phosphate                                                                     2.                                                       Enzyme-hydrolyzed casein.sup.1                                                                     10.                                                      Tap water            q.s. 1 liter                                             ______________________________________                                         .sup.1 "NZ Case," Sheffield Chemical Co., Norwich, N.Y.                  

This vegetative inoculum, in a 500-ml. flask, was shaken on a rotaryshaker at 250 rpm for 24-48 hours at a temperature of about 25°C.

Following the incubation of the vegetative inoculum, five percent of thevolume of the vegetative inoculum containging viable vegetative growthwas employed to inoculate the sterile fermentation medium for theproduction of the A-26771 antibiotics. The production medium has thefollowing composition:

    Ingredient         Percent                                                    ______________________________________                                        Glucose            4.0                                                        Molasses           2.0                                                        Peptone.sup.1      1.5                                                        Tap water          q.s. 25 liters                                             ______________________________________                                         .sup.1 Wilson's Meat Packing Co., No. 159                                

The pH of the medium thus prepared was 6.3; it was adjusted to pH 7.0with sodium hydroxide. The medium was then sterilized by autoclaving at120°C. for 30 minutes at 15-20 pounds per square inch pressure. Aftersterilization the pH was approximately 6. The inoculated productionmedium, contained in a 40-liter fermentor, was allowed to ferment at atemperature of about 25°C. for 6 to 7 days. The fermentation was aeratedwith sterile air at a rate of one-half volume of air per volume ofculture medium per minute.

At intervals during the fermentation, small samples of the productionmedium were withdrawn and assayed for antibiotic activity against one ormore of the following organisms: Sarcina lutea, Staphylococcus aureus,or Bacillus subtilis.

During the course of the fermentation, the pH of the medium graduallydecreased to a final value of between pH 3.6-4.2. Antibiotic A-26771factors A, B, and C are co-produced during the above-describedfermentation. Factors A and C are isolated from the filtered broth, andfactor B is isolated from the mycelia.

In Example 2 which follows, the separation, purification and isolationof the individual A-26771 antibiotics from the fermentation medium isdescribed. Because of the greater abundance of factor B in the A-26771mixture, it is the most easily isolated factor of the three thus faridentified in the A-26771 mixture.

EXAMPLE 2 Isolation of Factors A, B, and C

Factor B

The fermentation medium (100 liters) was filtered, using a filter aid.The mycelia were extracted with ethyl acetate (40 liters), and themycelial cake was discarded. The ethyl acetate phase was dried (Na₂ SO₄)and concentrated under vacuum to a volume of 4 liters. After cooling for72 hours at 4° C., a semicrystalline mass had precipitated. Thisprecipitate was separated by filtration, and the dark-colored filtratewas discarded. The precipitate (200 g.) was dissolved in ethyl acetate(2 liters) and filtered. The filtrate was chilled at 4° C. for 24 hoursto afford 46 g. of crystalline factor B. Three more crystallizations offactor B from the filtrate during the following days, using theprocedure described hereinabove, afforded a total of 110 g. ofcrystalline factor B, melting at 125° C. The properties of the fourcrystalline crops were identical in all respects.

Factor A

The filtered broth from 100-liters of fermentation medium, obtained asdescribed hereinabove, was extracted with an equal volume of ethylacetate. The ethyl acetate extract was dried (Na₂ SO₄) and concentratedto an oil under vacuum. This oil (15.4 g.) was chromatographed over a5.0 × 62.0 cm. column of silica gel (grade 62; 60-200 mesh; Matheson,Coleman and Bell). The column was eluted at a rate of 3 ml./min. withethyl acetate-methanol (9:1), 20-ml. fractions being collected.

During both this and subsequent chromatographies, the presence of factorA in column fractions was determined by subjecting the fractions tothin-layer chromatography on silica gel (E. Merck, 20 × 20 cm.precoated, F-254, layer thickness 0.25 mm. on glass, BrinkmannInstruments, Inc.), developing in benzene-methanol (95:5) and usingSarcina lutea as the bioautograph organism.

The active fractions were combined and concentrated in vacuo to an oilyresidue (1.8 g.). This residue was rechromatographed on a 4.5 × 39.0 cm.column of silica gel (grade 62, 60-200 mesh), eluting withbenzene-methanol (95:5) at a flow-rate of 3 ml./min.

The active fractions from this column were combined andrechromatographed on a 5.0 × 50.0 cm. column of silica gel (grade 62;60-200 mesh), eluting with chloroform at a flow-rate of 0.5 ml./min. andcollecting 5 ml.-fractions. Factor A was eluted in fractions 169-210.The fractions containing factor A were chromatographed a final time on a2.0 × 110.0 cm. column of silica gel (grade 62, 60-200 mesh), elutingwith a chloroform-methanol (98.5:1.5) solvent mixture. The activefractions from this chromatography were combined and yielded factor A(90 mg.) as yellow, prismatic crystals from acetone-diethylether-pentane melting at 105° C.

Factor C

During the chromatographic separations described hereinabove for factorA, the presence of another biologically active compound was observed.The fractions containing this compound were combined and concentratedunder vacuum to an oil. This oil was rechromatographed on a 2.0 × 100.0cm. column of silica gel (grade 62), eluting with an ethylacetate-hexane (1:2) solvent system at a flow-rate of 2 ml./min. Thefractions containing the new active material were combined andconcentrated under vacuum to an oil which crystallized fromacetone-diethyl ether-pentane to give 12 mg. of factor C, melting atabout 130° C.

The presence of factor C in column fractions was determined by thethin-layer chromatographic method described hereinabove for factor A. Inaddition, the antibiotic A-26771 factors can be characterized bythin-layer chromatography on silica gel (E. Merck, 20 × 20 cm.,precoated, F-254, layer thickness 0.25 mm. on glass, BrinkmannInstruments, Inc.) in a chloroform-methanol (98:2) solvent system withRf values as follows(detection using either ultraviolet absorption orsilver nitrate spray reagent number 224 as described in "Thin-LayerChromatography," 2nd ed., E. Stahl, Ed., Springer-Verlag, New York,N.Y., 1969, p. 897):

    Factor           Rf Values relative                                           ______________________________________                                        A                0.48                                                         B                0.05                                                         C                0.37                                                         ______________________________________                                    

EXAMPLE 3 Preparation of Factor B Methyl Ester

The methyl ester of A-26771 factor B has been prepared by methylationwith either a) 3% methanolic HCl or b) trimethyloxonium fluoborate inmethylene chloride, using standard procedures. The methyl estercrystallizes from methanol, melting point 90°-91° C. The infraredabsorption spectrum of the methyl ester (KBr) shows carbonyl bands at1745 (shoulder), 1735, 1718 and 1702 cm⁻ ¹. The mass spectrum showsmolecular ion at 396.21409, corroborating the 396.21480 theoretical massfor C₂₁ H₃₂ O₇.

We claim:
 1. The method for producing antibiotic A-26771 factors A, B,and C which comprises cultivating Penicillium turbatum NRRL 5630 in aculture medium containing assimilable sources of carbohydrate, nitrogen,and inorganic salts until a substantial amount of antibiotic activity isproduced by said organism in said culture medium, and separating theA-26771 antibiotic mixtures from said culture medium.
 2. The method ofclaim 1 which includes the additional step of isolating A-26771 factor Afrom said A-26771 antibiotic mixtures.
 3. The method of claim 1 whichincludes the additional step of isolating A-26771 factor B from saidA-26771 antibiotic mixtures.
 4. The method of claim 1 which includes theadditional step of isolating A-26771 factor C from said A-26771antibiotic mixtures.